ab120687
Pitstop 2™
Instructions for Use
This product is for research use only and is not
intended for therapeutic or diagnostic use.
1
Table of Contents
1.
Introduction
3
2.
Storage and Handling
4
3.
Preparation of Working Solutions
5
4.
Guidelines for in vitro use
6
2
1. Introduction
Pitstop 2™ directly binds to the clathrin terminal domain at a site that
overlaps with that of clathrin box containing accessory protein ligands
(von Kleist et al, 2011). In the case of amphiphysin 1 the IC50 value is
about 12 µM. However, this may vary for other proteins, typically between
10 and 60 µM. This is a medium-to-low affinity compound.
Pitstop 2™ is an amphiphile, which allows the compound to penetrate cell
membranes. As with all amphiphiles, it is advisable not to overdose the
application of the drug as this will likely lead to strong side-effects. Thus
users are advised to follow the protocol provided.
3
2. Storage and Handling

A stock solution should be prepared by dissolving in 100% DMSO
(fresh, sterile) to give a final concentration of 30 mM. Vortexing
should be used to ensure solublization of the compound. This
stock solution is stable at room temperature for about 4-6 hrs.

.Aliquots of the stock solution should be stored at -20°C. Stock
solutions should not be left at room temperature for longer than
necessary to prepare the working solutions. Avoid freeze/thaw
cycles of the stock solution as this may affect the activity of the
compound.

The final working concentration of DMSO should generally be
between 0.3 and 1%. Low concentrations of DMSO (i.e. 0.1%)
may lead the compound to precipitate out of solution. However,
please note that the amount of DMSO needed to keep the
compound in solution is dependent on the concentration of the
compound. For example, in 1% DMSO, drug concentrations of
300 µM or higher may also lead to precipitation (depending on
which aqueous buffer is used and the temperature).
4
3. Preparation of Working Solutions

Pitstop 2™ can be directly diluted in aqueous media to the final
working concentration and is compatible with a range of buffers.

Pitstop 2™ and the Pitstop 2™ negative control are not water
soluble, but can be used in low concentrations of DMSO (up to
1% for cellular experiments, up to 3% for in vitro use, see above
and below) after serial dilution from the 100% DMSO stock
solution.

Clathrin-mediated endocytosis (CME) is a highly cooperative
process; complete inhibition of CME at about 20-25 µM Pitstop
2™ in most cell types has been observed. A final working
concentration of 25 µM is therefore recommended. Most cell lines
will tolerate a concentration of 30 µM Pitstop 2™ but primary cells
generally tend to be more sensitive and could suffer from nonspecific damage.

The effect of storage and the re-use of the working solutions after
dilution in aqueous buffers has not been investigated and is not
recommended.
5
4. Guidelines for in vitro use
1. For enzymatic experiments, a 1 - 3% DMSO final concentration is
TM
possible as, in the experience of the inventors of Pitstop 2 , 3%
DMSO is the maximum tolerable concentration for most enzymes.
However, for best results, it is recommended to always perform a
DMSO dose titration to determine the maximum tolerable amount
of DMSO for the enzyme of interest. After this amount of DMSO is
determined, use this amount (or a sub maximum amount) to
solubilise the compound
2. For cell experiments cells should be grown to at least 80-90%
confluency on matrigel or poly-lysine coated supports. The media
should then be removed and a serum-free media used.
Pitstop 2™ is amphiphilic and, like many small molecules, is
sequestered by serum albumins. 0.1 - 0.2% serum can be
tolerated for some cell lines, however this has not been
extensively investigated. To the cells, add the working solution of
Pitstop 2™ dissolved in buffer or serum free media (containing 10
mM HEPES; pH 7.4) to give a final concentration of 25 µM (in up
to 1% DMSO). Incubate for 5 - 10 minutes, typically at 37°C. This
incubation period should be sufficient to completely block CME of
transferrin and other ligands, although the required time may
depend on the cell line and ligand
6
Longer incubation times (such as greater than 30 minutes) may
lead to non-specific effects and are therefore not recommended.
Use of final concentrations of 30 µM or above may occasionally
lead to cells lifting from the plates (certain cell lines).
Additionally, high concentrations of Pitstop 2™ may interfere with
fluorescence imaging unless cells are fixed. This fluorescence is
not usually detectable if the cells have been first fixed and washed
prior to imaging.
Special note when using electrically excitable cells, e.g. neurons: Neurons
are particularly sensitive to amphiphilic compounds and may respond by
firing action potentials. A final concentration of 15 µM Pitstop 2™ is
sufficient
to
completely
block
compensatory
endocytosis
at
the
presynaptic compartment.
A great advantage of Pitstop 2™ is that its effects on endocytosis are
reversible. Incubation of Pitstop 2™-treated cells for 45-60 min (with two
changes of media) in full serum containing medium will restore the ability
of cells to undergo CME. This is an important control experiment that may
be included in all experimental trials.
For technical questions please do not hesitate to contact us by email
(technical@abcam.com) or phone (select “contact us” on
www.abcam.com for the phone number for your region).
7
8
9
10
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226)
Canada
Email: ca.technical@abcam.com | Tel: 877-749-8807
China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
11